Megalomicin and methods for production thereof

ABSTRACT

A NEW ANTIBIOTIC COMPLEX IDENTIFIED LAS ANTIBOTIC W847 COMPLEX (OR ANTIBIOTIC W847 OR MEGALOMICIN), COMPOSED OF AT LEAST FOUR MAJOR ANTIBIOTIC COMPONENTS IS PRODUCED BY THE CULTIVATION UNDER SUBMERGED AREOBIC CONDITIONS OF MICROMONOSPORA MEGALOMICEA, A HIHERTO UNDESCRIBED SPECIES OF THE GENUS MICROMONOSPORA OF THE ORDER ACTINOMYCETALES. THE INDIVIDUAL COMPONENTS ARE ISOLATED BY SOLVENT EXTRACTION AND CHROMATOGRAPHY AND ARE CHARACTERIZED BY THEIR RESPECTIVE CHEMICAL AND BIOLOGICAL PROPERTIES.

4, 1972 M. J. WEINSTEIN ETA-l. 3,632,750

MEGALOMICIN AND METHODS FOR PRODUCTION THEREOF 3 Sheets-Sheet 1 Filed Sept.

9 I0 WAVELENGTH IMICRONSI FIG. 1

INFRARED SPECTRUM 0F ANTIBIOTIC W84 7-A 7 WAVELENGTH (MICRONS) INFRARED SPECTRUM OF ANTIBIOTIC W847- B 0 O O 3 Q m 8 6 4 2 3L woz tzm N MN A M M :G SAM m mw W n LMW w N w vmfl/p. WW I I A 4 WE V .1 M TZ. W E WU B EQ w Lw M .IAVI W I E I) F Ms m o .w 0 M C .wm v P R I 4 w N W m w G m N DT E E0 L Rm mm A F W W A 5 4 u I 3 G Jan. 4, 1972 J w Ns ETAL 3,632,750

MEGALOMICIN AND METHODS FOR PRODUCTION THEREOF Filed Sept. 4, 1969 3 SheetsSheet 3 4000 3000 2000 I500 I I000 900 800 700 I00 TRANSMITTANCE (70) 4 INFRARED SPECTRUM OF ANTIBIOTIC W847- C 7.0 6T0 5:0 4:0 3:0 2:0 L0 NUCLEAR MAGNETIC RESONANCE SPECTRUM 5 OF ANTIBIOTIC w 47- A NUCLEAR MAGNETIC RESONANCE SPECTRUM 6 OF ANTIBIOTIC W847 B INVENTORS' MARVIN J.WEINSTEIN,GERALD H. WAGMAN JOSEPH A.MARQUEZ,GEORGE M. LUEDEMANN Jan. 4, 1972 J w ms m ETAL 3,632,750

MEGALOMICIN AND METHODS FOR PRODUCTION THEREOF Filed Sept. 4, 1969 I5 Sheets-Sheet 5 Bio 760 6:0 510 4.0 3 0 2.0 LO 0 NUCLEAR MAGNETIC RESONANCE SPECTRUM OF ANTIBIOTIC W847-C FIG. 7

8.0 7:0 6:0 5:0 4:0 3:0 2:0 ITO NUCLEAR MAGNETIC RESONANCE SPECTRUM OF ANTIBIOTIC W847-C FIG. 8

INVENTORS MARVIN J.WEINSTE!N,GERALD H.WAGMAN JOSEPH AMARQUEZJSEORGE M. LUEDEMANN BY B65 19 AGENTS United States Patent 3,632,750 MEGALOMICIN AND METHODS FOR PRODUCTION THEREOF Marvin J. Weinstein, East Brunswick, George M. Luedemann, Glen Ridge, Gerald H. Wagman, East Brunswick, and Joseph A. Marquez, Montclair, NJ-, assrgnors to Schering Corporation, Bloomfield, NJ. Continuation-impart of application Ser. No. 707,100, Feb. 21, 1968, which is a continuation-in-part of application Ser. No. 641,522, May 26, 1967. This application Sept. 4, 1969, Ser. No. 855,424

Int. Cl. A61k 21/00 US. Cl. 424-120 17 Claims ABSTRACT OF THE DISCLOSURE A new antibiotic complex identified as Antibiotic W847 Complex (or Antibiotic W847 or megalomicin), composed of at least four major antibiotic components is produced by the cultivation under submerged areobic conditions of Micromonospora megalomicea, a hitherto undescribed species of the genus Micromonospora of the order Actinomycetales. The individual components are isolated by solvent extraction and chromatography and are characterized by their respective chemical and biological properties.

FIELD OF THE INVENTION This invention is a continuation-in-part of our copending application Ser. No. 707, 100, filed Feb. 21, 1968 now abandoned, which is a continuation-in-part of application Ser. No. 641,522, filed May 26, 1967 (now abandoned).

This invention relates to new antibiotics and to methods for their production, isolation, and purification. More specifically, this invention relates to new and useful antibiotics formed by the cultivation under controlled conditions of a hitherto undescribed species of the genus Micromonospora of the order Actinomycetales.

DESCRIPTION OF THE INVENTION The microorganism The hitherto undescribed species of the genus Micromonospora employed in the production of the new antibiotics of this invention was isolated originally from a soil sample obtained directly from the earth by one of the inventors named herein. Two strains of this Micromonospora species have been isolated. These two strains are natural color variants and may be distinguished microscopically by the degree to which sporulation occurs. Cultures of living organisms of these two strains have been deposited and made a part of the stock culture collection of the US. Department of Agriculture, Northern Utilization Research and Development Division, Peoria, 111., where they have been designated NRRL 3274 and NR-RL 3275. These strains are available from the collection agency upon request. These strains will be referred to hereinafter individually as Micromonospora sp. W847 (NRRL 3274) and as Micromonospora sp. W847 (NRRL 3275) and collectively as Micromonospora sp. W847. These strains are also referred to by applicants as Micromonospora megalomicea var. megalomicen NRRL 3274 and Micromonospora megalomicea var. nigra NRRL 3275, respectively These names are deemed to be synonymous with those set forth above and may be used interchangeably.

Macroscopic examination of 28-day old cultures (24- 26 C.) of Micromonospora sp. W847 (NRRL 3274) on Czapeks sucrose agar is characterized by good growth exhibiting no aerial mycelium and by raised and plicate Patented Jan. 4, 1972 colonies of a soft, moist and non-waxy consistency produclng no diiiusable pigment. The color of the colony surface, described according to the Descriptive Color Name Dictionary, Taylor, Knoche and Granville, published by the Container Corporation of America, 1950 (U.S.A.), together with the color chip number taken from the Color Harmony Manual, 4th edition, 1958, published by the same company, is orange m4-la. This color corresponds to the synonym or near synonym, strong orange 50, as found in the National Bureau of Standards Circular No. 553, Nov. 1, 1955, U.S.A. (all colony color designations employed hereafter are based upon the same color reference system).

Microscopic examination of the same culture shows a regular mycelium composed of long branched filaments approximately 0.6g in diameter. Spores are infrequently found.

Macroscopic examination of 28-day old cultures of Micromonospora sp. W847 (NRRL 3275), grown on the same medium and under the same conditions described above, is characterized by good growth exhibiting the same gross colony characteristics as the previous strain except for color. The color of the colony surface is black Microscopically, this culture shows a regular mycelium composed of long branched filaments approximately 0.6 1. in diameter. In this strain, however, spores are very abundant and are randomly produced throughout the mycelium. The spores are ovoid to spherical in shape with a 0.7-1.0,u. diameter and are dark brown when mature.

Colonies of Micromonospora sp. W847 may be further characterized by their growth characteristics on various nutrient media. Table 1 below gives the growth characteristics of Micromonospora sp. W847 on some of the more widely used nutrient media. The colony observations were made on 14-day old (24-26 C.) cultures unless otherwise specified.

TABLE I Colony characteristics of Micromonospora sp. W847 on various media Glucose asparagine agar.-Growth poor.

Aerobic or anaerobic.-Aerobic.

Bennetts agar.--Growth poor.

Emersons agar.Growth poor.

Tomato paste-oatmeal agar.--Growth poor.

Glucose-yeast extract -agar.-Growth good, raised, finely plicate.

Potato slice.Growth poor.

Sucrose-nitrate agar (Czapeks agar).-Growth good,

raised, plicate.

Tyrosine agar (Observations at 2, 7, and 14 days (after Gordon and Smith, I. Bact. 69:147)).Growth fair, crystals poorly dissolved directly under colony, brown ditfusible, pigment produced after 3 weeks, diffusible pigment not black or typical of a tyrosine positive as produced by streptomyces.

Micromonospora sp. W847 shows good growth at temperatures between 26 and 37 C. and no growth at 50 C. It is aerobic and does not reduce nitrate to nitrite. This species does not liquify gelatin, does not hydrolize milk and does not decompose cellulose. No diffusible pigment is produced on peptone-iron agar.

Micromonospora sp. W847 shows good growth on carbohydrate media containing 0.5 percent yeast extract base and 1 percent of l-arabinose, glucose, starch, and sucrose but only fair growth where the carbohydrate source is 1 percent xylose. It grows poorly, if at all, on such media in which the sole carbohydrate source is 1 percent darabinose, cellulose, dulcitol, galactose, glycerol, lactose, levulose, melibiose, melizitose, raflinose, rhamnose, ribose, inositol or mannitol.

Further characteristic of Micromonospora sp. W847 is its ability to utilize nitrogen sources. Colony growth char acteristics on typical nitrogen sources, observed after 14 days (24-26" C.) growth, are set forth in Table 2 below.

TABLE 2 Utilization of nitrogen sources Nitogen source plus 1% glucose: Growth characteristics.

0.5% Difco yeast extract Growth good, raised,

finely plicate.

1.0% NZ amine Type A 1 Growth fair. 1% asparagine Growth poor. 1% gultamic acid Do. 1% ammonium nitrate Do.

1 Product of Sheffield Chemical Company, Norwich NY.

The antibiotic hereinafter to be described is prepared through utilization of Micromonospora sp. W847 var. NRRL 3274 and var. NRRL 3275. The instant invention, however, is not to be construed as so limited as to exclude the use of other variants of Micromonospora sp. W847 or mutants thereof produced from this organism by mutating agents such as, for example, high frequency radia tion including X-ray and ultraviolet, actinophages and nitrogen mustard. In the following description wherein the process for antibiotic production is elaborated, it is to be understood that the use of the term, Micromonospora sp. W847, is intended to include the color variants specifically described above as well as all other fully equivalent variants and mutants of this species.

THE ANTIBIOTICS (1) Preparation of antibiotic W847 complex Micromonospora sp. W847, by controlled fermentation techniques as described hereafter, produces a complex mixture of antibiotic substances hereinafter referred to as the Aantibiotic W847 complex. This antibiotic complex may also be referred to as Antibiotic W847 or megalomicin; the names are synonymous and may be used interchangeably.

In order to prepare a suitable inoculum for subsequent fermentation, Micromonospora sp. W847 is grown under submerged aerobic conditions with continual agitation at temperatures ranging from about 22 to about 37 C. for a period of from about 2 to about 4 days. Generally a temperature of 35 C. over a 3-day period is found most satisfactory. The medium normally used in this first ger mination stage is aqueous and contains quantities of carbon and nitrogen sources assimilable by the organism. The alkalinity of the media desirably is adjusted to about pH 7.5 before autoclaving by the addition of base such as sodium hydroxide.

Exemplary of media employed in the germination stage, are the following: Medium 1: Bacto-beef extract (Difco), 3.0 gm.; Bacto-tryptone (Difco), 5.0 gm.; dextrose, 1.0 gm.; starch (potato), 24.0 gm.; Bacto-yeast extract (Difco), 5.0 gm.; CaCO 2.0 gm.; tap water, 1000 ml.; Medium 2: Bacto-yeast extract (Difco), 5.0 gm.; fish solubles, 1.0 gm.; corn steep liquor, 1.0 gm.; dextrose, 20.0 gm.; CaCO 1.0 gm.; tap water, 1000 ml.

Desirably, a second germination stage (e.g. inoculum stage) is employed prior to fermentation and harvesting of the antibiotic in order to enrich the inoculum. Employing media as described above, fresh medium is inoculated with the germination culture previously prepared w./v.) and incubated at about 22 to about 37 C. for about 2 to about 4 days. Usually, a temperature of 28 C. over a period of 3 days is found most satisfactory.

In order to produce the Antibiotic W847 complex, a 5 percent (w./v.) inoculum taken from the inoculum stage is added to aqueous fermentation medium which, prior to autoclaving, has been adjusted to about pH 7.1 to 7.8. Air usually about 3 to about 6 liters per minute, is

introduced into the fermentation which, generally, is conducted at a temperature between about 22 to about 37 C. over a period of about 2 to about 6 days with continuous agitation. Particularly with reference to Medium 3, described below, it has been found that maximum yields of Antibiotic W847 complex are obtained following fermentation for 66 hours at 31 C. with introduction of air at a rate of 4.5 liters per minute and with impeller agitation at 250 r.p.m. Using 14-liter fermenters containing 10 liters of medium, yields ranging from 300 to 800 meg/ml. have been obtained under these conditions.

Media useful in the fermentation stage include, for example, the following: Medium 3: sucrose, 30.0 gm.; dextrose, 2.5 gm.; trypticase, 17.0 gm.; NaNO 3.0 gm.; K HPO 3.5 gm.; MgSO -7H O, 0.5 gm.; KCl, 0.5 gm.; FeSO 0.01 gm.; NaCl, 5.0 gm.; tap water, 1000 ml.; Medium 4: Bacto-yeast extract (Difco), 5.0 gm.; dextrose, 10.0 gm.; starch, 20.0 gm.; casein hydrolysate, 5.0 gm.; CaCO 4.0 gm.; tap water, 1000 ml.; Medium 5: sucrose 20.0 gm.; peptone, 5.0 gm.; cotton seed protein hydrolysate, 5.0 gm.; CaCO 4.0 gm.; tap water, 1000 ml.; Medium 6: sucrose, 30.0 gm.; peptone, 8.0 gm.; NaNO 3.0 gm.; K HPO 1.0 gm.; MgSO -7H O, 0.5 gm.; FeSO 0.01 gm.; tap water, 1000 ml.; Medium 7; trypticase, 17 gm.; NaCl, 5.0 gm.; K HPO 2.5 gm.; dextrose, 2.5 gm.; Czapek-Dox broth, 35.0 gm.; tap water, q.s. 1000 ml.

For recovery of Antibiotic W847 complex from the fermentation medium, the whole broth is adjusted to about pH 9.5 with base such as, for example, sodium hydroxide. The whole broth then is extracted with about 2 volumes for each volume of broth with a suitable immiscible organic solvent, such as, for example, ethyl acetate, methylene dichloride, butyl acetate, chloroform,

butanol and the like. The solvent phase is next concentrated under vacuum to a small volume, approximately 100 mold concentration, and is purified by column chromatography employing, for example, LH 20 Sephadex, an alkylated cross-linked dextran (Pharmacia Fine Chemicals, Inc., Uppsala, Sweden) suspended in about percent aqueous ethanol. The column is eluted with the aqueous ethanol. Column fractions are combined according to their antibacterial activities by agar disc testing against Staphylococcus aureus (ATCC 6538P), concentrated to dryness, dissolved in a small amount of ethyl ether or acetone and added to an excess volume of petroleum ether (B.P. about 30 to about 60 C.). The mixture is then transferred to a Dry Ice-acetone bath (approximately 50 C.) and allowed to stand for about 20 minutes after which it is allowed to return to room temperature. The mother liquor is separated from any oily residue by decanting and is concentrated to dryness. Material produced in this manner is usually found to have potency of about 232 mcg./mg. according to the bio-assay procedure described below.

The assay is a cylinder cup assay using Sarcz'na Zutea (ATCC 9341) as the test organism. The conditions of the assay are similar to the cylinder plate assay described for erythromycin (Grove and Randall, 1955, Med. Encyclopedia, Inc., N.Y.). A mcg. of Antibiotic W847 complex activity is the amount of material which produces a zonal response of 20.8:05 mm. under the conditions of this assay.

Alternatively, Antibiotic W847 complex may be prepared by utilizing the following fermentation and isolation techniques:

Employing Medium 1 for the preparation of an inoculum, fermentation flasks containing this medium are inoculated with a culture (approximately 5% w./v.) of Micromonospora sp. W847. The flasks are placed on a rotary shaker running at about 280' strokes per minute and are incubated at 28 C. for 72 hours. At the end of this time, fermentors, each charged with fermentation medium (Medium 7 for example), adjusted to pH 7.15 to 7.25, are inoculated with approximately 5% (v./v.) of the 72 hour inoculum culture. The fermentation to the agar. The paper and silica gel are removed after 15 minutes. The agar plate is incubated at 37 C. over night and the zones of inhibition evaluated.

Results of these experiments are shown in Table 4 below.

TABLE 4.COMPARATIVE THIN LAYER CHROMATOGRAPHY OF ANTIBIOTIC W847 COMPLEX R: of

inhibition System Antibiotic R: and color by H2804 spray zone Chioroform:methanol:17% ammonia 2:1:1 (v./v.) Antibiotic W847 complex Blue black.... 0. 98 0.98 Oleandomycin Green 0. 98 0. 98

Erythromycin. Yellow tan 0. 98 0. 98

Magnamycin Blue purple 0. 98 0.98

Butanohacetic acidzwater 3:1:1 (v./v.) Antibiotic W847 complex Red purple 0.13 0.13 Erythromycim Green brown. 0. 26 0. 26

Spiramyclm Purple; gray- 0. 06; 30 0. 06

Oleandomycin. Green 0.19 0. 19

Magnamycin Purple; red purple 0.40; 0. 47 0. 40

after 24 hours and to 700 r.p.m. after 48 hours. Fermentation is terminated at the end of about '69 hours. The broth from all fermentors is pooled for extraction and recovery of Antibiotic W847 complex as described below.

The pooled fermentation broth is adjusted to about pH 9.25 with aqueous sodium hydroxide (50%) and is extracted with about 2 volumes of ethyl acetate and the extracts are concentrated to about one to two percent of the original volume. The ethyl acetate concentrate is then extracted at least twice with half volumes of hydrochloric acid (about 0.14 N). Alternatively, the acid extraction can be carried out with other mineral acids at about the same normality. The combined acid extracts are made slightly alkaline with aqueous sodium hydroxide (to about pH 8.5 to 9.0) and are extracted at least twice with about equal volumes of ethyl acetate. The combined ethyl acetate extracts are concentrated to dryness under reduced pressure to obtain the Antibiotic W847 complex. The complex at this stage will show a potency of 450 to 550 meg/mg. in the assay described above.

(2) Chemical characterization of Antibiotic W847 complex Antibiotic W847 complex was compared by paper chromatography with a variety of antibiotics, including several macrolides. In a series of solvent systems, as shown in Table 3 below, Antibiotic W847 complex moves as a single spot and is differentiated from all other antibiotics except the macrolides which were tested (i.e. magnamycin, oleandomycin, spiramycin and erythromycin).

Further, by comparison of the physical, chemical and biological properties of Antibiotic W847 complex, and of its separated fractions, as hereinafter described with properties of known macrolides as reported in the literature, Antibiotic W847 complex and its components are distinguishable from all known macrolide antibiotics.

Antibiotic W847 complex gives a positive color reaction in the Ninhydrin, Starch-KI and Biuret tests and a negative color reaction in the Stannous Chloride, Molisch and Sa-kaguchi tests. The complex is essentially transparent in the ultraviolet region. The activity of Antibiotic W847 complex is not significantly altered when a solution thereof is subjected to a temperature of 100 C. for 30 minutes throughout the pH range of 2 to 10 (stability is tested by dissolving the antibiotic in ethanol, diluting with buffer, and disc testing against S. aureus and Escherichia coli). The complex shows no loss in activity after treatment with trypsin, chymotrypsin, pepsin, a-amylase or penicillinase in buffered solutions (optimal pH for each enzyme) at 37 C. for up to 24 hours exposure. Antibiotic W847 complex is soluble in most polar organic solvents such as, for example, ethyl acetate, methanol, ethanol and acetone.

(3) Separation of Antibiotic W847 components Bioautography of thin layer silica gel plates on agar seeded with Sarcina lutea following chromatography in a solvent system consisting of chloroformzmethanol (:40 v./v.) indicates that Antibiotic W847 complex consists of at least four antibiotic components which will be identified hereafter as Antibiotic W847 Fractions A, B, C and TABLE 3.COMPARAT1VE Ri VALUES OF ANTIBIOTIC W847 COMPLEX Ris of antibiotics Antibiotic Chlor- Chlor- Paper chromatographic W847 Magna- Oleando-Ethryro- Spira- Pem- Kana-amphentetra- Genta- Strepto- Tetrasystems complex mycin mycin mycm mycin cilhn myciu icol cyeline micin mycin cycline 807ffl1ncighanol/pipi 37sodiuan c or e w.v. escening* E 0. 90 0. 90 0. 90 0. 90 0. 90 0. 63 0. 30 0. 84 0, 74 0, 59 0 57 0, 7 Propanol: pyridine: acetic acidzwater (6:421:15) ascending (v./v.) 0. 93 0. 93 0. 93 0.93 0. 93 0. 91 0.18 0. 91 0. 75 0.26 0. 40 0.70 Butanol acetic acid :water (4:1:5) ascending (v./v.) 0.78 0. 78 0. 78 0. 73 0.78 0.85 0.00 0. 92 0. 46 0.00 0.00 0. 38 80% phenol ascending (v./v.) 0.86 0.86 0.86 0.86 0.86 0.81 0.17 0.93 0.87 0.50 p 0.06 0.83

*Paper buffered with 0.95 molar NazSO4-l-0.05 molar N aHSOr.

Differentiation of Antibiotic W847 complex from these important macrolide antibiotics was made by thin layer chromatography on silica gel GF plates (Analtech Inc., Wilmington, Del.). Zones were detected by the following two methods:

(1) Plates are sparyed with a mixture of concentrated sulfuric acid-methanol (1:1 v./v.) and developed by heating at 105 C. for several minutes. The zones of organic material appear as colored areas against a white background.

(2) Plates are allowed to remain in contact with an agar layer seeded with S. aureus. A sheet of whatman No. 1 paper is placed between the seeded agar layer and C (or as Antibiotic W847-A, Antibiotic W847-B, Antibiotic W847-C and Antibiotic W847-C These fractions may also be referred to as megalomicin-A, magalomicin-B, megalomicin-C and megalomicin-c respectively. These fractions, in the solvent system described above, show Rf values of 0.19 (Fraction A), 0.38 (Fraction B), 0.52 (Fraction C and 0.65 (Fraction C respectively.

Separation of the fractions is achieved by dissolving Antibiotic W847 complex in acetone mixed with silicic acid, evaporating the acetone and adding the dry mixture to the top of a silicic acid column. The column is eluted with chloroformimethanol (60:40 v./v.) and fractions are the plate to insure that the silica gel does not adhere collected. The column is monitored by disc testing each fraction against S. aureus and E. coli. The active fractions are chromatographed on silica gel thin layer plates run for about 1.5 hours in the same solvent system used for the column. The patterns of the antibiotic fractions are detected by the two methods previously described. Fractions are combined according to their chromatographic patterns and are concentrated.

By this method, Fraction C as a hydrochloride salt is separated and isolated as a White powder by dissolving the pooled active fractions in methanol and precipitating with about volumes of ethyl ether. Fraction C hydrochloride at this stage exhibits a potency of about 1100 mcg./ mg.

During chromatography W847-C base is converted to a hydrochloride salt by reaction with small quantities of hydrogen chloride present in the solvent. The hydrochloride, as would be expected, does not exhibit appreciable solubility in ethyl ether and is, therefore, precipitated by the addition thereof.

The free base form of W847-C may be obtained by adding dilute alkali such as .1 N NaOH to an aqueous solution of the hydrochloride to about pH 8.5-9.5 and separating the free base by filtration from the aqueous suspension.

Later fractions collected from the column described above when pooled and concentrated to dryness yield a white powder Antibiotic W847-C fraction. At this stage, the Antibiotic W847-C powder obtained displays a potency of about 880 meg/mg. in the previously described cylinder cup assay.

Using the bioautograph techniques previously described (see Chemical Characterization of Antibiotic W847 Complex) and chloroform:methanol (60:40 v./v.) as the solvent system, the Antibiotic W847C isolate has broad spectrum antibacterial activity in vitro against the following gram-positive and gram-negative organisms:

Bacillus subtz'lis ATCC 6633 Sarcina lutea ATCC 9341 Staphylococcus aureus ATCC 65381 Pseudomonas aeruginosa ATCC 8689 The remaining active fractions from the silicic acid column are combined and further resolution is achieved by Florisil (magnesium silicate) column chromatography.

meg/mg. and 71 meg/mg. respectively, in the S. lutea assay.

Alternatively, a mixture of Antibiotic W847-C fractions may be isolated from the ethyl acetate concentrate prepared as described hereinabove. The residue is dissolved in acetone and poured into cold water With vigorous stirring. The suspension is permitted to warm to room temperature and filtered. The precipitate comprises a mixture of Antibiotic W847-C and Antibiotic W847-C said mixture sometimes referred to as Antibiotic W847-C complex (or Antibiotic W847-C or megalomicin-C). As precipitated, megalomiciu-C is in the form of the free base. An acid addition salt of the complex may conveniently be prepared by the titration of a methanol solution of the base with the appropriate acid and by precipitating the salt by the addition ofethyl ether. When the foregoing procedure is utilized to obtain the Antibiotic W847-C complex, Antibiotics W847-A and W847-B may be extracted from the aqueous filtrate and the extract subsequently concentrated to a residue and chromatographed on magnesium siilcate as described above.

(4) Chemical characterization of Antibiotic W847 Fractions A, B, C and C The antibiotics of this invention are nitrogen bases; consequently, each fraction is capable of forming acid addition salts in a manner similar to that described for the Antibiotic W847-C complex. Typical of such salts are those formed with such acids as hydrochloric, sulfuric, phosphoric, stearic, propionic, tartaric, maleic and the like. These salts display antibiotic activity similar to that of the antibiotic free base but may differ somewhat in potency and solubility characteristics.

The antibiotics of this invention are also hydroxylated compounds; consequently, they are capable of being esterified, preferably with a hydrocarbon carboxylic acid. Exemplary of such esters are acetates, propionates, cyclopropyl carboxylates, succinates, benzoates and the like. Such esters generally exhibit antibiotic activity similar to that of the free antibiotic usually differing in potency and solubility characteristics.

In Table 5 below is set forth the chemical and physical properties of Antibiotic W847 Fractions A, B, C and C TABLE 5.CHEMIOAL AND PHYSICAL PROPERTIES OF ANTIBIOTIC W847 FRACTIONS Fraction A Fraction B Fraction Ci Fraction O2 Optical-rotation lulu 1% in ethanol (deg.).. 1 92 104 102 Melting Point, C. (dec.) 255-259 1 125-135 243-246 146-150 PKa- 9.1 8.8 as 8.6 Neutrahzation Eq.. 442 490 488 492 Found Calculated Found Calculated Found Calculated Found Calculated Elemental analysis:

Carbon" 60. 23 60. 25 68. 90 60.11 60. 23 59. 98 58. 93 60. 35 Hydrogen 9. 38 9. 19 8. 94 8. 99 8. 93 8.81 8.73 8. 89 Nitrogem... 3.30 3.19 2.88 3.05 2. 94 2.92 2. 88 2.07 Oxygen (by dinerence)- 27.19 27. 36 29.18 27.85 27. 90 2s. 30 2 46 27. so

Empirical formula C44 soN2 1s C4s s2 2 1e C4B 84 2 l7 cn saNz n Molecular Weight 8 919. 14 961. 17 975. 20

1 Mouohydrate.

The column is prepared by activating the Florisil at 100 The infrared spectra of Antibiotic W847 Fractions A,

C. for 16 hours; fixing in hexane and pouring the resulting slurry into a glass column. The column is charged with residual Antibiotic W847 complex (after removal of Fractions C and C dissolved in methylene chloride and is developed progressively with one hold-up volume (i.e. that volume of solvent which is held by the absorbent) of hexane, one hold-up volume of ethyl ether, one hold-up volume of ethyl acetate and then with increasing amounts of acetone in ethyl acetate. Separation is monitored by disc testing the fractions against S. aureus and E. coli followed by thin layer chromatography as previously described.

Employing this technique, separation of Fraction A and Fraction B is accomplished. Both are isolated as off-white powders by concentration of the pooled chromatographic fractions to dryness. Fractions A and B assayed at 145 B, C and C are shown in the drawings as FIGS. 1, 2, 3 and 4 respectively. The spectra of the respective antibiotic fractions were run in mineral oil (Nujol) and the more significant absorption peaks are set forth in Table 6 with the following designations: S=strong, M=moderate, W: weak, VS=very strong, MS=moderate to strong, brd.= broad, shp.=sharp, shd.=shoulder and s.b.'=side band. The Antibiotic W847 Fractions A, B, C and C have characteristic nuclear magnetic resonance spectra which are shown in the drawings as FIGS. 5, 6, 7 and 8 respectively. The 'NMR spectra were observed on a Varian A60-A spectrometer on a solution (ca. 0.4 ml., ca. 20 mg./ml.) of the sample of each fraction in deuterated chloroform. The spectra are recorded in parts per million (p.p.m.) from tetramethylsilane, the internal standard.

TABLE 6.INFRARED SPECTRA F ANTIBIOTIC W847 FRACTIONS Fraction A Fraction B Fraction O1 Fractio C2 Wavc- Peak Wave- Peak Wave- Peak Wave- Peak Iength(u) strength length strength length strength length strength 2.92-2.90 MS,brd. 2.87 M 2.82 s.b. 2.83 W-M 3.55-3.50 Nujol 2.97 shd. 2.87 W 3 33348 Nujol 5.77 S 3.35-3.50 Nujol 3.35-3.50 Nujol 5.70 S 5.82 5.72 S 5.72 S 5.80 5.88 M-S 5.83 S 5.87 W-M 5.88 slid- 6.82 Nujol 6.82 Nu ol 6.82 Nujol 6.80 Nujol 7.24 Nu ol 7.25 Nujol 7.25 Nujol 7.23 Nujol 7.36 s.b. 8.05 7.45 M 8.00 S 7.82 M-S, 8.45 S,brd. 7.57 M 8.53 S,brd. 8.45 VS, brd 8.59 S 7.82 M 8.93 S 8.92 VS 8.94 S 8.03 S 9.08 51111. 9.02 VS 9.30 S 8.13 VS 9.27 M-S 9.12 VS 9.62 S 8.60 S 9.65 S 9.32 M-S 9.91 M-S 8.94 M-S 10.00 MS,brd. 9.53 VS 10.02 M-S 9.05 S 10.38 M 9.63 Vs 10.29 M 9.56 S 10.97 M 9.98 VS 10.84 W-M 9.70 S 11.15 M 10.27 S 11.08 W-M 10.13 M-S 11.52 W 11.02 M-S 11.20 W-M 10.48 M 11.92 W-M 11.17 M-S 11.02 .WM 11.18 W-M 1 Instrument artifact.

On the basis of the foregoing chemical and physical properties, Antibiotic W847 (megalomicin) is believed to have a gross molecular structure as follows:

OH; CH;

110 K]- I on. H01 J HO 0 0 on 3 M 0 3 no on.

0 g on. on,

Antibiotic W847-B (megalomicin-B), R =hydrogen,

R =acetyl Antibiotic W847-C (megalomicin-C R =acetyl Antibiotic W847-C (megal0micin-C R 1=acetyl,

R =propionyl (5 Biological properties of Antibiotic W847 complex, Fractions A, B, C C and Antibiotic W847-C complex (A) In vitro tests.-Antibiotic W847 complex, Fractions A, B, C and C and Antibiotic W847-C complex all possess a broad spectrum antibacterial activity in vitro against a variety of gram-positive and gram-negative organisms. Either as a complex or as individual fractions, therefore, they may be used to clean and sterilize laboratory glassware, to clean and sterilize surgical instruments and the like. Also, these antibiotics may be used in combination with soaps and detergents to clean and sanitize areas used for food preparation such as kitchens, dining halls and the like.

The in vitro antibacterial spectrum of Antibiotic W847 complex, Fractions A, B, C and C and Antibiotic W847-C complex against typical gram-positive and gram-negative organisms is shown in Table 7 below.

TABLE 7 Antibacterial spectrum of antibiotic W847 complex and fractions A, B, 01, C2 and C-complex Minimal inhibitory concentration (mag/m1.) Antibiotic W847 Complex Complex Fraction Fractio Fractio Fraction Microorganism base HCI A B 01 C2 C-complex Bacillus megatherium DA 7064 0. 3 0. 3 0. 6 1. 2 0. 6 0. 3 Bacillus subtilis ATCC 6633 0.03 0. 3 0. 005 0. 05 0.005 0.005 12', DA700 1.2 1.2 0.6 Diplococcus pneumoniae ATCC 10015- 0.5 0.5 0. 05 Il, DA 150 6.0 6.0 27 27 12.0 27 27 Euterococcus sp. DA 800 0. 3 0. 0. 6 0. 6 0. 3 0. 08 0. 3 Enteracoccux sp. DA 801 0. 3 0.75 0.6 0.6 0. 3 0. 6 0. 3 Enterococcus sp. DA 802 0.3 1. 0 0.2 0.2 0. 3 0. 03 0. 3 Sarcina lutea ATCC 9341 0. 0075 0075 0. 005 0. 005 0- 0075 0. 0005 0. 00075 Staphylococcus aureus ATCC 12715- 0.3 0.75 0.08 0. 2 0. 3 0. 005 0.03 Staphylococcus aureus ATCO 6538P- 0. 3 0.03 0. 6 O. 2 0. 03 0.005 0.03 Staphylococcus aureus ATCC 11631 0. 3 0. 075 0. 6 0. 6 0. 3 0. 6 0. 3 Staphylococcus aureus (Gray) 0.3 0. 3 0.6 0. 6 0. 3 0. 6 0. 5 Staphylococcus aurcus DA 2001"--- 0. 03 0. 3 0. 6 0. 2 0.03 0.03 0.03 Staphylococcus aurcus DA 2003 0.03 0. 03 0. 6 0.6 0. 075 0.6 0.3 Staphylococcus aureus DA 2010-.-" 0. 3 0. 8 0. 6 0. 6 0. 3 0. 2 0. 25 Staphylococcus aureus DA 2014 0. 3 0.3 0.6 0. 6 0. 03 0. 6 0.03 Staphylococcus aureus DA 2018 0. 3 0. 3 0. 6 0. 6 0.075 0. 1 0. 1 Staphylococcus aureus DA 2032- 0. 075 0. 3 0.6 0. 6 0.075 0.08 0. 08 Staphylococcus aureus DA 2033 L... 16 16 2. 7 2. 7 32. 0 2. 7 2. 7 Streptococcus faecalis DA 20 0. 3 0- 3 0- 0 0. 1 0. 3 0.005 0. 075 7 DA, 21 0. 75 10 5.0 5.0 0.3 0.6 0. 3 Streptococcus pyoyenes DA 11 0.3 0. 6 0.2 0. 3 0.03 0. 3 Streptococcus pyoye'aes DA 12 0. 0. 2 0.03 Mycobacterium smegmatis ATOC 1 0143 0. 75 0. 3 5. 0 5. 0 0.3 0.05 0. 3 Escherichia coli ATCC 10536 6.0 16 6. 0 12.0 12. 0 12. 0 12. 0 Klebsiclla pneumom'ae ATCC 10031.. 12. 0 16 6. 0 24. 0 12. 0 24. 0 12. 0 Proteus vulgaris DA 121 6. 0 16 12. 0 24.0 24. 0 24. 0 24. 0 Pseudomonas aerugiuosa ATCC 8689- 6.0 12. 0 6. 0 12. 0 6. 0 6. 0 6. 0 Salmonella schottmuelleri DA 10 6. 0 12. 0 6. 0 12. 0 12. 0 12. 0 12. 0

1 Penicillin resistant strain. I 2 Erythromycin resistant strain.

Norm-Medium: Yeast beet broth (pH 8.0). Comp1ex=Mixture of components A, B, C; and Cg- 11 The susceptibility of the test organisms to the antibiotics was determined by a tube dilution assay in yeast beef broth adjusted to pH 8.0 with sodium hydroxide.

The antibacterial activity of Antibiotic W847 complex is compared with that of erythromycin, penicillin G and methicillin in Table 8 below (susceptibility being determined by the tube dilution assay technique described above). The results show that Antibiotic W847 complex compares favorably in activity to the other antibiotics tested against gram-positive bacteria. In addition, Antibiotic W847 complex is active against Mycobacterium smegmatis at a lower concentration than erythromycin, while penicillin G and methicillin are inactive up to 10 meg/ml.

TABLE 8 organisms including species of the genus Escherichia, Salmonella, Proteus and Pseudomonas. These organisms are responsible for many serious disease syndromes including urinary tract infections and diarrheas. Such syndromes are quite common in domestic animals such as cattle, horses, sheep, swine, dogs and cats and may be effectively controlled and treated by means of the antibiotics described herein.

Antibiotic W847 complex suspended in peanut oil gives an LD (lethal dose for 50 percent of the population tested) of 270 mg./kg. in mice by the intraperitoneal route. On subcutaneous and oral administration, the LD is greater than 5000 mg./kg. The PD (protective dose for 50 percent of the population tested) is 150 mg./kg.

Comparison of the antibacterial spectrum of Antibiotic W847 complex with erythromycin, penicillin G and methieillin Penicillin G Methicillin 1 Clinical isolates including penicillin resistant organisms. 2 Clinical isolates resistant to erythromycin.

No'rE.-Med.ium: Antibiotic W847 complex and erythromycin in yeast beef broth at pH 8.0. Penicillin G and methieillin in yeast beet broth at pH 6.8.

(B) In vivo tests.As Was noted above, Antibiotic W847 complex, Antibiotic W847-C complex, Fractions A, B, C and C exhibit a broad range of anti-microbial activity against gram-positive and gram-negative microorganisms. Included in the gram positive group are pathogenic microorganisms including species of the genus Streptococcus, Staphylococcus and Diplococcus which are known to cause many disease manifestations. Various species of Staphylococcus and Streptococcus are the organisms responsible for causing bovine mastitis. These species are readily controlled and treated by means of the antibiotics disclosed herein after 'a relatively brief regimen of administration.

Antibiotic W847 complex, Fractions A, B, C C and W847-C complex are also active against gram-negative by the subcutaneous route (peanut oil suspension) against a lethal infection of S. aureus in mice. Against Streptococcus pyogenes by the same route, the PD in mice is mg./ kg.

In addition to the foregoing, Antibiotic W847 complex administered at 17.5 mg./kg. to dogs is detectable in the blood serum for 7 days. This is surprising in view of the fact that when a comparable dose of erythromycin is given it does not persist throughout a 24 hour interval.

Acute toxicity data for Antibiotic W847 fractions and complexes are compared with erythromycin in Table 10 below.

Tables 11, 12 and 13 are set forth to illustrate the animal protection exhibited by the antibiotics of the instant invention.

TABLE 10 Toxicity of Antibiotic W847 fractions and complexes and of erythromycin in mice (LD50 in mgJkg.)

Erythrocornplex complex W847 Fraction B Inycin base Erythrobase in HCl in Fraction A base in W847-C W847-C1 W847-C2 in peanut mycin HCl Route peanut oil H O base 1 peanut oil complex 1 base 1 HCl in H2O oil in H2O Oral 1, 2, 500 7, 500 500 1, 000 500 2 3, 000 2 2, 927 Subcutaueous 1, 160 2, 500 7, 000 500 500 1, 000 500 2, 500 2 1, 849 1ntrapcrito11cal it 75 300 350 400 500 500 590 2 400 1 In 0.5% aqueous carboxymethyl cellulose.

3 Values reported by Grundy (1966) Experimental Chemotherapy, vol. 111.

TABLE 11 Activity of Antibiotic W847 complex and erythromycin against lethal intraperitoncal infections in mice (IDso in mgJkg.)

Erythromyoin Antibiotic Antibiotic Antibiotic W847 Fraction Base in W847 complex W847 complex C2 H01 in peanut H01 Microorganism in peanut oil HCl in H H20 oil in H O Subcutaneous treatment Staphylococcus aureus Gray Streptococcus pyoaenes DA 21 Diplococcus pneumoniae DA 150 1 PDmo 2 Per day.

TABLE 12 Activity of Antibiotic W847 fractions and ery thi'omyciu against lethal iutraperitioneal infections in mice (PDao in mg./kg.)

Treatment W847-A W847-O1 Erythro- Microorganism route base 1 base 1 mycin base 1 Streptococcus pyoyeues C 203 Oral 300 207 147 Do S.C. 220 118 134 Streptococcus pyogenes O Oral 300 300 180 Do 5.0. 134 180 Streptococcus pyoyeues No. 22 ral 180 250 Do S.C. 180 200 50 Streptococcus pyogenes No. 9. Oral 250 250 Do- SC. 167 250 Staphylococcus aureus Smith ral 250 180 167 o- 3.0. 75 117 40 Staphylococcus aureus Gray Oral 300 250 106 D0- .C 20 62 30 Staphylococcus aureus W- 117 53 Do- 20 25 20 Staphylococcus aureus No. 41-... 757 D0- 20 25 20 Dip eye. 800 300 134 D0 90 167 90 Diplococcus p N o. 2 250 200 134 Do 70 170 75 Euterococcus No. 802 300 250 300 Do SC. 160 180 Euterococous N o. 804 Oral 250 250 250 Do. 3.0. 180 250 250 Escherichia colt Oral 200 250 250 Do 8.0. 100 250 160 Klebsz'ella Oral 250 250 250 Do- 8.0. 150 250 250 Pseudomoncs aerugirtosa Oral 250 250 250 Do-.. 3.0. 161 250 130 1 In 0.5% aqueous carboxymethylcellulose. Z S.O.=subcutaneous.

TABLE 13 Activity of Antibiotic W847 fractions and combinations thereof against lethal intraperitoneal infections in mice (PDso 1n mg./kg.)

W847-C W847-B W847-C2 W847-C complex base in W847-C1 HCl in complex H01 in peanut oil base 1 H2O base 1 H20 Subcutaneous administration of antibiotics:

Organism:

Staphylococcus aureus Gray 150 200 45 50 50 Streptococcus pyoyeues C 150 10 50 50 Staphylococcus aureus Smith 86 Streptococcus pyoyeues No. 5 39 Oral administration of antibiotics:

Organism:

Straphylococcus aureus Gray 250 100 390 250 Streptococcus pyoyencs C 250 100 310 250 Straphylococcus aureus Smith 310 Streptococcus pyogeues No. 5 390 1 Ultrasonicated suspension in aqueous 0.5% carboxymethyl cellulose.

Antitiotic W847 Fraction A suspended in 0.5% aqueous carboxymethylcellulose and dispersed by ultrasonication is active by subcutaneous administration in mice against S. aureus Gray with a PD (protective dose for 50 percent of the population tested) of 20 mg./kg. and against P aeruginosa with a PD of 161 mg./kg. The LD in mice by the subcutaneous route is 7000 mg./ kg.

Antibiotic W847 Fraction B suspended in peanut oil is active by subcutaneous administration in mice against S. aureus with a PD of 150 mg./kg. and against P. aertcginosa, 350 mg./kg. delays death by 24 hours. The LD in mice by the subcutaneous route is greater than 500 rug/kg.

Antibiotic W847 Fraction C suspended in 0.5% aqueous carboxymethyl cellulose and dispersed by ultrasonication is active in mice against S. aureus. When administered subcutaneously against S. aureus Gray has a PD of 62 mg./kg. By the same route it has a PD of 180 ing/kg. against S. pyogerzes C. The antibiotic is somewhat less active orally having a PD against S. aureus Gray and S. pyogenes C of greater than 250 mg./ kg. The LD in mice by the subcutaneous route is greater than 1000 mg./kg. Antibiotic W847-C exhibits antimalarial activity against Plasmodium berghei and when administered to rodents 100 mg./ kg./ day) 24 hours postinfection causes a significant suppression in the blood parasite count.

Antibiotic W847 Fraction C as a suspension in aqueous carboxymethyl cellulose is active by subcutaneous administration. It is, however, more advantageously used as an aqueous solution of its hydrochloride salt and when administered subcutaneously to mice has a PD of 45 mg./kg. against S. aureus Gray and a PD of mg./kg. against S. pyogenes C. By the oral route it has a PD of 100 mg./kg. against both organisms. The LD in mice by intraperitoneal administration is greater than 500 rug/kg.

PREPARATION OF PHYSICAL EMBODIMENTS The following examples will more fully illustrate the preparation of the antibiotics described above and constitute the best mode contemplated by applicants for carrying out their invention.

(1) Fermentation of the microorganism Example 1.Shake flask fermentation (A) Germination stage.Add a 0.5 ml. lyophilized culture of Micromonospora sp. W847 to a 300 ml. Erlenmeyer flask containing 100 ml. of the following medium which has been adjusted to pH 7.5 with dilute sodium hydroxide prior to sterilization:

Tap water, 1000 ml.

Incubate the flask and its contents for 3 days at 37 C. on a rotary shaker (280 r.p.m.; 2-inch stroke).

(B) Inoculum stage.Aseptically transfer a 25 ml. inoculum from the germination stage to a 2 liter Erlenmeyer flask containing 500 ml. of the sterile medium used above for germination. Incubate the flask and its contents for 3 days at 28 C. on a rotary shaker (280 r.p.m.; 2-inch stroke).

(C) Fermentation stage.--Aseptically transfer 25 ml. aliquots from the inoculum stage to 2-liter Erlenmeyer flasks containing 500 ml. of the following sterile medium which has been adjusted to pH 7.8 with dilute aqueous sodium hydroxide prior to sterilization.

Gm. Sucrose 30.0 Sodium nitrate 3.0 Dipotassium phosphate 1.0 Magnesium sulfate 0.5 Ferrous sulfate 0.01

Peptone 8.0

Tap water, 1000 ml.

test organism. The concentrate will give a zone of inhibition of approximately 20-35 mm.

Example 2.Tank fermentation Temperature-3 1 C.

Sterile air input-4 liters/minute Agitation-250 r.p.m.

Anti-foam (Dow Corning B)add as needed At the end of this period, the potency of the produced antibiotic reaches a peak which remains substantially constant. Throughout fermentation, the pH of the fermentation mixture remains substantially in the range of 7.2 to 8.2. The packed cell volume reaches a constant value of 3.5-4.5 ml. The activity of the produced anti biotic in the whole broth gives a zone diameter of l525 mm. when disc tested against S. aureus or P. aeruginosa.

Example 3.Tank fermentation (A) Preparation of inoculum.-To four 2-liter flasks each containing .500 ml. of sterile broth as described in Example 1A, add 0.5 ml. of a lyophilized culture of Micromonospora sp. W847. Place the flasks on a rotary shaker running at 280 strokes per minute (2-inch stroke) and incubate at 28 C. for 72 hours.

(B) Fermentation stage.To each of four fermentors, add 10 liters of the following sterile production medium adjusted to pH 7.15 to 7.25 prior to sterilization:

Gm. Trypticase 170.0 Sodium chloride 50.0 Dipotassium phosphate 25.0 Dextrose 25.0 Czapek-Dox broth 350.0

G.E.-60 Antifoam (General Electric Company,

Schenectady, N.Y.). Tap water, q.s. 10 liters.

Inoculate each fermentor with 500 ml. of the 72 hour seed culture prepared above. Bring the temperature of the fermentation medium to 31 C. and agitate at 500 r.p.m. while introducing air flow through the medium at the rate of 0.5 liter of air/liter of broth/minute. Increase the agitation rate to 600 r.p.m. after 24 hours and to 700 r.p.m. after 48 hours. Terminate the fermentation at the end of 69 hours. Pool the broths from the four fermentors for extraction (about 37 liters).

(2) Extraction of Antibiotic W847 complex Example 4.Extraction of Antibiotic W847 complex from laboratory fermentation (chromatographic technique) Adjust 60 liters of whole broth prepared according to the procedure described in Example 2 to pH 9.5 with dilute aqueous sodium hydroxide. Extract with 2 volumes of ethyl acetate for each volume of broth. Separate the solvent phase and concentrate under vacuum to obtain an oily residue (approximately 30.0 g.). The biological activity (1/20 dilution) of this oily residue will give a zone of inhibition diameter of about 20-30 mm. against S. am'eus and about 15-25 mm. against P. aeruginosa. Purify the oily residue by column chromatography according to the following technique:

Prepare a column using 1000 g. of LHZO Sephadex (Pharmacia Fine Chemical, Inc.) suspended in percent aqueous ethanol. Transfer the oily residue to the column and elute with 95 percent ethanol at a flow rate of 200 17 mL/hr. Collect 50 ml. fractions. Combine fractions according to their antibacterial activity (determined by agar disc testing against S. aureus ATC'C 65 38P). Concentrate fractions having peak activity to dryness. Dissolve the solid residue in a small amount of acetone and pour into an excess volume of petroleum ether (B.P. 30-60 C.). Transfer the mixture to a Dry Ice-acetone bath (approximately 50 C.) and allow to stand 20 minutes. Allow the mixture to return to room temperature and separate the mother liquor from the oily residue by decanting. Concentrate the mother liquor to dryness to obtain purified Antibiotic W847 complex.

Antibiotic W847 complex so produced will have a potency of about 225 to 275 meg/mg. when assayed according to the cylinder cup technique described above.

Example 5. Extraction of Antibiotic W847 complex from tank fermentation (solvent extraction technique) Adjust the pooled broth (about 37 liters) obtained as described in Example 3 to pH 9.5 with 50' percent aqueous sodium hydroxide. Extract with two volumes of ethyl acetate and evaporate the extract to a volume of 2,150 ml. Isolate the Antibiotic W847 complex by one of the following techniques:

(A) Hydrochloric acid extraction.Extract a 500-ml. aliquot of the ethyl acetate concentrate twice with 250 ml. portions of 0.5 percent (0.14 N) hydrochloric acid. Make the combined acid extracts slightly alkaline by the addition of 5 percent aqueous sodium hydroxide (about pH 8.5 to 9.0) and extract twice with 250 ml. portions of ethyl acetate. Combine the ethyl acetate extracts and concentrate under reduced pressure to dryness. The complex so produced will have a potency of about 525 mcg./ mg. when assayed by the previously described cylinder cup technique.

(B) Sulfuric acid extraction-Repeat the extraction described in Example 5A substituting 0.1 N sulfuric acid for the hydrochloric acid. The complex so produced will have a potency of about 5.50 meg/mg. in the cylinder cup assay.

(3) Resolution of antibiotic W847 complex Example 6.- Isolation of Antibiotic W847 Fraction C Prepare a column (105 cm. long, 7.6 cm. diameter) using 2000 g. of silicic acid (Analytical Reagent, Mallinckrodt) by packing small segments into a compact bed. Dissolve 13.0 g. of Antibiotic W847 complex as prepared in Example 4 in acetone mixed with silicic acid and evaporate the acetone under vacuum. Add the dry mixture to the top of the column. Elute the column with a mixture of chloroform (60 parts by volume) and methanol (40 parts by volume) at a flow rate of 100 mL/hr. Collect 200 ml. fractions. Monitor'the column by disc testing each fraction against S. aureus and E. 0011'. Chromatograph the active fractions on silicic acid thin layer plates run for 1.5 hours in the same solvent system used for the column. Combine fractions according to their chromatographic patterns are determined by the two methods previously described (see Chemical Characterization of Antibiotic W847 Complex). Concentrate the fractions under vacuum. By combining fractions as indicated in Table 14 below, Antibiotic W847 Fraction C hydrochloride is isolated as a white powder by dissolving the combined residues in one volume of methanol and precipitating with volumes of ethyl ether. This material at this stage has a potency of about 1000 mcg./mg. in the cylinder cup assay previously described.

Isolation of Antibiotic W847 Fraction C material By combining later fractions as indicated in Table 14 below, an Antibiotic W847 Fraction C isolate is obtained as a white powder by concentrating the selected column fractions to dryness. At this stage, this material exhibits a potency of about 900 mcg./ mg. in the cylinder cup assay previously described. The remaining active material is eluted from the column, pooled and concentrated to a residue.

Example 7.-Isolation of Antibiotic W847 Fraction A and Fraction B Activate 500 g. of Florisil at C. for 16 hours; fix in hexane and slurry into a glass column cm. long; 3.8 cm. diameter; hold-up volume 70 0 ml.). Charge the column with 2.8 g. of the residue from Example 6, dissolved in 10 ml. of methylene chloride. Elute the column progressively with 700* m1. of hexane, 700 ml. of ether, 700 ml. of ethyl acetate and increasing amounts of acetone in ethyl acetate (flow rate 200 ml./hr.). Collect 100' ml. fractions. The column is monitored as previously described by testing fractions against S. aureus and E. coli followed by thin layer chromatography. Combine the fractions as indicated in Table 14 below.

TABLE 14.-RESULTS OF COLUMN SEPARATION Thin layer pattern R; chloroformmethanol (60/40) Potency Fraction Column cut Weight (meg/mg.)

Siiicic acid column 28.48 mg u 850 1000 Florisil column W847B 1 164-227 W847A 2 370-397 1 30% acetone in ethyl acetate. 2 80% acetone in ethyl acetate.

Suspend about mg. of Antibiotic W847 complex, as isolated in Example 5 above, in about 50 ml. of water. Adjust the pH of the suspension to about 6.5 to 7.0 by the addition of dilute (1 N) hydrochloric acid and stir vigorously. Remove the insolubles, if any remain, by filtration and lyophilize the filtrate to obtain an Antibiotic W847 hydrochloride weighing approximately 90 mgs. and assaying about 550 mcg./mg.

By the utilization of substantially the procedure of this example on either the Antibiotic W847 complex or the various fractions thereof and by substituting equivalent quantities of such acids as sulfuric, phosphoric and the like, their respective acid addition salts may also be prepared. Applicants consider all such salts to be the full equivalent of the antibiotics described herein and to fall within the scope of their invention.

Table 15 below sets forth the activity of the hydrochloride, sulfate and phosphate salts of Antibiotic W847 complex against a variety of gram-positive and gram-negative organisms.

TABLE 15 Antibacterial spectrum of acid addition water soluble salts of Antibiotic W847 complex Minimum inhibitory concentration mcg./ml.)

Hydrochloride salt Sulfate Phosphate salt salt Microorganism NorE.-Medium: Yeast beef broth pH 8.0. Salts made from Antibiotic W847 complex with a potency of 232 meg/mg.

19 Example 9.-Isolation of Antibiotic W847C complex Dissolve 542 grams of W847 complex (see Example in 5.4 liters of acetone and treat the resulting solution with 81 grams of decolorizing carbon for about 30 minutes at about room temperature. Remove the decolorizing carbon by filtration and concentrate the filtrate in vacuo to approximately 2.0 liters. Prepare a slurry of about 80 liters of ice and water and with vigorous stirring add the acetone solution. Allow the temperature of the resulting suspension to rise to about 25 C. with stirring and collect the product by filtration. Wash the solids with a small quantity of water and dry at about 50 C. in vacuo to yield about 212 grams of Antibiotic W847-C complex assaying about 1075 meg/mg.

Additional product (about 85 grams) may be obtained by concentration of the mother liquor, however, this product may be contaminated with Fractions A and B.

Antibiotic W847-C complex as prepared above may advantageously be used in the preparation of acid addition salts as shown below.

Example l0.--Preparation of a tartrate salt of an Antibiotic W847-C complex To a solid composition comprising 2.0 g. of a complex of Antibiotic W847-C and Antibiotic W847-C obtained in Example 9 above, add 30 ml. of diethyl ether with stirring. Add to the resulting solution 0.5 g. of tartaric acid dissolved in 2.5 ml. of ethanol. Stir the mixture for about minutes, then filter the precipitated salt. Wash the precipitate with diethyl ether and dry the product to obtain about 2.0 g. of tartrate salt whose bioassay is about 575 mcg./mg. and whose rotation is (57) concentration 1% in ethanol.

Example 11.-Antibiotic W847-C complex propionate Dissolve 1.0 g. of Antibiotic W847-C complex in 5.0 ml. of dry acetone and add 0.35 ml. of propionic anhydride. Stir the mixture at room temptrature for 4 hours, then slowly and with stirring add 8 ml. of 1.2% ammonium hydroxide solution. Filter the resulting precipitate and wash with 40% aqueous acetone, then with water to obtain the desired product. The product obtained in this manner has the following constants: M.P. 207- 210 C.; [a] =-63 (C.=1) ethanol.

Example 12.-Antibiotic W847-C complex diacetate Dissolve 11.0 g. of W847-C complex in 55 ml. of pyridine and add 5.5 ml. of acetic anhydride. Stir the reaction mixture at room temperature for about 18 hours, then pour it into 2.0 l. of ice water containing 50 ml. of 14% ammonium hydroxide with vigorous stirring. Filter the resulting precipitate, wash with water and dry to yield the desired product melting 237241 C. and having a rotation of about 86, C.-=l in ethanol.

Alternatively, the precipitated product may be extracted with ethyl acetate, the extract Washed with water and evaporated in vacuo to a residue. The residue is subsequently dissolved in acetone, treated with decolorizing carbon and precipitated by the addition of an equal volume of water to yield the product of this example.

Example 13.Antibiotic W847-C diacetate Dissolve 10.0 g. of Antibiotic W847-C in 50 ml. of pyridine; add 5.0 ml. of acetic anhydride and keep the mixture at room temperature for about 18 hours. Pour the solution into 2 liters of ice water and add 50 ml. of 14% ammonium hydroxide. Filter the resulting suspension, wash the precipitate with water and dry. Crystallize the precipitate from aqueous acetone, treating with decolorizing carbon if needed, to obtain the title product, M.P. 240-243" C.,[a] =86 (C.=1, ethanol).

20 Example 14. Antibi0tic W847-C dipropionate Dissolve 10.0 g. of Antibiotic W847-C in 50 ml. of pyridine; add 5.0 ml. of propionic anhydride and hold the resulting solution at room temperature for about 18 hours. Pour the solution into 2 liters of ice water and add 50 ml. of 14% ammonium hydroxide. Extract the product with methylene chloride and wash the extract with water. Concentrate the extract to a residue and crystallize the residue from aqueous acetone to obtain the product of this example, M.P. 174177 C., [a] =84- (C.=1, ethanol).

Example 15.Antibiotic W847B diacetate Example 16.Antibiotic W847-A tripropionate Dissolve 10.0 g. of Antibiotic W847-A in 200 ml. of pyridine; add 20.0 ml. of propionic anhydride and hold the mixture at room temperature for about 18 hours. By the isolation method of Example 15, there is obtained the product of this example, M.P. l77l79 C.,

lD (ethanol).

Example 17.-Antibiotic W847-A dipropionate To a solution of 1.0 g. of Antibiotic W847-A, add 0.5 ml. of propionic anhydride. Stir the solution at room temperature for 24 hours, then dilute with 1.2% ammonium hydroxide with stirring. Filter the resulting ssupension, Wash with dilute aqueous acetone then with water and dry. Crystallize from aqueous acetone to obtain the compound of this example, M.P. 102108 C., [a] =72 (C.=l, ethanol).

Example 18.-Antibiotic W847A dibenzoate Suspend 250 mg. of Antibiotic W847-A and 150 mg. of sodium bicarbonate in 4 ml. of acetone. Add mg. of benzoyl chloride and stir the mixture at room temperature for about 4 hours and filter the mixture. Concentrate the filtrate under reduced pressure to a residue. Dissolve the residue in about 5 ml. of acetone and dilute slowly with 5% ammonium hydroxide. Filter the resulting precipitate, wash with water and dry to obtain the product of this example, M.P. 222-225 C.

Example 19.--Antibiotic W847-A tribenzoate Dissolve 250 mg. of Antibiotic W847-A in 5 ml. of pyridine and add 0.14 ml. of benzoyl chloride with stirring. To keep the solution at room temperature overnight, then concentrate under reduced pressure to about 1 ml. and dilute with about 5 ml. of acetone. Add 5% ammonium hydroxide until the product precipitates. Filter the suspension, wash the precipitate with water and dry to obtain the product of this example, M.P. 144149 C.

Example 20.Antibiotic W847-C dibenzoate Esterify Antibiotic W847-C with benzoyl chloride according to the procedure of Example 18. Isolate the product as described and obtain the title product, M.P. 166- 169 C.

Example 21.Antibiotic W847-A triacetate To a solution of 10.0 g. of megalomicin-A (from Ex ample 7) in 200 ml. of pyridine add 20.0 ml. of acetic 21 anhydride and keep the reaction mixture at room temperature for 18 hours. Work up the reaction mixture according to the procedure described in Example 13. Crystallize the product from aqueous acetone to obtain the compound of this example, M.P. 199202 C.,

By substituting an equivalent quantity of isovaleric anhydride for the acetic anhydride in the preceding example and by following the procedure of said example. Antibiotic W847-A tri-isovalerate is obtained.

Similarly, by substituting an equivalent quantity of stearoyl chloride for the acetic anhydride in Example 21 and by following the procedure of said example, Antibiotic W847-A tristearate is obtained.

Example 22.-Antibiotic W847-A triacetate lauryl sulfate Dissolve 250 mg. of Antibiotic W847-A triacetate, from the preceding example, in 3 ml. of acetone and add a solution of sodium lauryl sulfate in 3 ml. of water. Add with stirring a mixture of 0.03 ml. of acetic acid and 0.2 ml. of water. Dilute the mixture to a volume of 30 ml. with water and refrigerate overnight. Decant the supernatant and dissolve the waxy residue in acetone. Pour the acetone solution into ether, filter the resulting suspension and dry the precipitate at room temperature to obtain the compound of this example, M.P. 135-40 C.

Example 23.Megalomicin-A hemisuccinate To 1.0 g. of megalomicin-A in 20 ml. of acetone add 228 mg. of succinic anhydride. Heat the mixture to boiling and remove any insoluble material by filtration. Heat the filtrate briefly at refiux, then allow to stand at room temperature for 18 hours. Dissolve the oily precipitate by warming the mixture, concentrate to one half volume, add a small amount of methanol and pour slowly with agitation into 200 ml. of ether. Filter the resulting precipitate, wash with ether and dry to obtain a product which TLC and bioautography shows to consist of two active components. Separate by chromatography on silica gel to obtain megalomicin-A di-hemisuccinate, M.P. 163 166 C. and megalomicin-A tri-hemisuccinate, M.P. 165 167 C.

In view of the foregoing examples, it is apparent that the antibiotics of this invention will form esters with a host of other acyl residues and applicants consider all such esters to be the full equivalent of those whose preparation is described herein.

The following examples are directed to some of the pharmaceutical dosage forms wherein the antibiotics may be used. The formulations, excluding the topicals, are designed to permit the administration of from about 5 to about 50 mg. of antibiotic per kilogram of body weight per day. It is contemplated that the medicaments be given in doses up to 8 times in a 24 hour period.

The topical formulations are generally applied to the infected area from 2 to 4 times daily.

In the utilization of the formulations set forth below the dosage of anti-biotic given and the frequency of administration depends in large part on the stage and severity of the infection, the type of infection and the individual characteristics of the animal species being treated.

22 Procedure:

(1) Blend the Antibiotic W847 complex and the lactOSC (2) Add the magnesium stearate and mix (3) Fill capsules Sorbitol solution, U.S.P 500.0

Water, q.s. 1000 ml.

Procedure 1) Heat 200 ml. of water to boiling, and dissolve in it one half of the parabens. Cool to about 70 C., then mix in the Polysorbate 80. Sprinkle in the silicate, stirring until a uniform smooth suspension results.

(2) Heat an additional 200 ml. of water to boiling, and dissolve in it the remainder of the parabens. Disperse the CMC in this until a smooth gel results. Mix in the sorbitol solution. Then dissolve the sodium citrate.

(3) Add (2) to (1) slowly, with constant stirring. Cool the mixture to 25 C. Add the Antibiotic W847A, flavor and color, mixing thoroughly. Add sufficient quantity of water to make the total volume 1000 ml.

Example 26 Topical cream: Gm.

Antibiotic W847B 10 Stearic acid 200 Sorbitan monostearate 104 Sorbitan monooleate 20 Polyoxyethylene sorbitan monolaurate 56 Water, q.s. 1000 gm.

Procedure (1) Heat the stearic acid, sorbitan monostearate, sorbitan monooleate, and polyoxyethylene sorbitan monolaurate to 65 C.

(2) Heat about of the water to 70 C.

(3) Add the water to Step 1 and mix to form the cream base.

(4) Slurry the Antibiotic W847B with about 10% of the water and pass through a colloid mill.

(5) Add the milled slurry to the molten base and mix. Allow to cool.

Example 27 Topical ointment: Gm. Antibiotic W847-C 10 Petrolatum 990 Procedure (1) Melt the petrolatum.

(2) -Slurry the Antibiotic W847 -C with about 10% of the petrolatum and pass through a colloid mill.

(3) Mix the milled slurry with the remainder of the molten petrolatum. Allow to cool.

23 Example 28 Parenteral suspension: Mg. Antibiotic W847-C 250 Sodium carboxymethylcellulose 3 'Polysorbate 80, U.S.P. l Methylparaben 3.6 Propylparaben 0.4

Water, q.s. 2.0 ml.

Procedure (1) Prepare a solution of sodium carboxymethylccllulose, Polysorbate 80, U.S.P., methylparaben, and propylparaben. Autoclave the solution.

(2) Aseptically, slurry sterile Antibiotic W847-C with a portion of the above vehicle and pass through a colloid mill.

(3) Mix the milled slurry with the remainder of the vehicle.

(4) Fill into sterile vials.

We claim:

1. A method for preparing megalomicin which comprises cultivating a member selected from the group consisting of M icromonospora megalomicea var. megalomicea NRRL 3274 and Micromonospora megalomicea var. nigra NRRL 3275 in an aqueous nutrient medium under submerged aerobic conditions until substantial antibiotic activity is produced.

2. The method of claim 1 including the step of recovering megalomicin which comprises rendering the whole broth alkaline, extracting with an immiscible solvent and isolating megalomicin from said solvent.

3. The method of claim 2 including further the step of separating megalomicin into its component fractions which comprises absorbing megalomicin atop a chromatographic column and selectively desorbing megalomicin- C megalomicin-C megalomicin-B, and megalomicin-A from said column.

4. Megalomicin by the process of claim 1 having the following characteristics:

(a) exhibits a positive color reaction in each of the ninhydrin, starch KI and biuret tests,

(b) exhibits no color reaction in each of the stannous chloride, Molisch and Sakaguchi tests,

() possesses an antibacterial spectrum substantially as shown in Table 7 with substantially no diminution in potency after contact for 24 hours at 37 C. with any of the enzymes, trypsin, chymotrypsin, pepsin, u-amylase and penicillinase; and

((1) consists of at least four antibiotic substances, megalomicin-A, megalomicin-B, megalomicin-C and megalomicin-C 5. A composition of matter selected from the group consisting of megalomicin-A, megalomicin-B, megalomicin-C megalomicin-C and the non-toxic pharmaceutically acceptable acid addition salts thereof wherein:

(1) megalomicin-A is an organic substance having the following characteristics:

(a) Empirical formula: C H N O (b) Melting point: 255 -259 C. dec.

(0) Ultra violet spectrum (95% ethanol): essentially transparent (d) Optical rotation (measured by the D line of sodium at C. in ethanol at 1% concentration): 90

(e) pKa: 9.1

(f) Neutralization eq.: 442

(g) Infrared spectrum essentially as shown in FIG.

1 with major peaks as set forth in Table 6;

(h) Nuclear magnetic resonance spectrum: as

shown in FIG. 5.

(i) Antibacterial spectrum substantially as shown in Table 7 24 (2) megalomicin-B is an organic substance having the following characteristics:

(a) Empirical formula: C46H32N2O16 (b) Melting point: 125l35 C. (c) Ultra violet spectrum ethanol): essentially transparent (d) Optical rotation (measured by the D line of sodium at 25 C. in ethanol at 1% concentration): 92 (e) pKa: 8.8 (f) Neutralization eq.: 490 (g) Infrared spectrum essentially as shown in FIG.

2 with major peaks as set forth in Table 6 (h) Nuclear magnetic resonance spectrum: as

shown in FIG. 6 (i) Antibacterial spectrum substantially as shown in Table 7; (3) megalomicin-C is an organic substance having the following characteristics:

(a) Empirical formula: C H N O (b) Melting point: 243246 C. dec. (c) Ultra violet spectrum (95% ethanol): essentially transparent (d) Optical rotation (measured by the D line of sodium at 25 C. in ethanol): 104 (e) pKa: 8.8 (f) Neutralization eq.: 488 (g) Infrared spectrum essentially as shown in FIG.

3 with major peaks as set forth in Table 6 (h) Nuclear magnetic resonance spectrum: as

shown in FIG. 7 (i) Antibacterial spectrum substantially as shown in Table 7; (4) megalomicin-C is an organic substance having the following characteristics:

(a) Empirical formula: C H N O (b) Melting point: l46-150 C. dec. (c) Ultra violet spectrum (95% ethanol): essentially transparent (d) Optical rotation (measured by the D line of sodium at 25 C. in ethanol at 1% concentration): -102 (e) pKa: 8.6 (f) Neutralization eq.: 492 (g) Infrared spectrum essentially as shown in FIG.

4 with major peaks as set forth in Table 6 (h) Nuclear magnetic resonance spectrum: as

shown in FIG. 8 (i) Antibacterial spectrum substantially as shown in Table 7.

6. A composition of claim 5, said composition being megalomicin-A.

7. A composition of claim 5, said composition being megalomicin-B.

8. A composition of claim 5, said composition being megalomicin-C 9. A composition of claim 5, said composition being megalomicin-C 10. A composition of matter of claim 5, said composition being a non-toxic pharmaceutically acceptable acid addition salt of megalomicin-A.

11. A composition of matter of claim 5, said composition being a non-toxic pharmaceutically acceptable acid addition salt of megalomicin-B.

12. A composition of matter of claim 5, said composition being a non-toxic pharmaceutically acceptable acid addition salt of megalomicin-C 13. A composition of matter of claim 5, said composition being a non-toxic pharmaceutically acceptable acid addition salt of megalomicin-C 14. A composition of matter of claim 13, said composition being megalomicin-C hydrochloride.

15. A composition of matter of claim 10, said composition being megalomicin-A hydrochloride.

25 26 16. A composition of matter of claim 10, said comties, McGraw-Hill Book Co., Inc., New York, Jan. 25, position being megalomicin-A tartrate. 1962, pp. 595 and 596 (N0. 1203).

17. A non-toxic pharmaceutically acceptable acid addition alt of megalomicin of cl i 4, JEROME D. GOLDBERG, Primary Examiner 5 References Cited U.S. Cl. X.R.

Miller, the Pfizer Handbook of Microbial Metaboli- 424-121; 195-450 

